Pharmacopoeia Case Study

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2.1. Plant materials Sijunzi Decoction (SJZD) was composed of Codonopsis pilosula, Atractylodes macrocephala, Poria cocos and Glycyrrhizae uralensis, and these four drugs were in same ratio as Pharmacopoeia. Dried herbs were purchased from Kangmei Pharmaceutical Company Ltd. (Guangzhou, Guangdong, China). 2.2. Reagents DMEM medium, fetal bovine serum (FBS) and NEAA were purchased from GIBCO Laboratories (Grand Island, NY, USA). MTT and phenolsulfonphthalein were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Annexin V-FITC/PI was from Lianke Biotechnology Co (Hangzhou, China). The anti-claudin 1, anti-claudin 2, anti-zo3, anti-occludin antibody, anti- myosin light chain (phosphor S20) antibody, anti- myosin light chain antibody, anti-myosin…show more content…
Next, the herbal drugs were added with water of about 5 times the amount of the drugs, then decocted and boiled for 30 min and filtered again. Then the two filtrates were mixed together and concentrated to 1.4 L, we added ethanol to filtrates as the alcohol concentration of 75%, put them in a refrigerator at 4 ℃ overnight, filtrated, precipitated and dissolved filtrates in about 1.6 L ultrapure water, then centrifugated (8400 rpm, 15 min), the supernatant was frozen and dried to gain the total polysaccharide; phenol-sulfuric acid method by spectrophotometry meter was used to measure the content of polysaccharide (as glucose), which was 70.61±1.70% (at least three independent experiments). 2.4. Cell culture The human colon adenocarcinoma cell line Caco2, purchased from the Cell Culture Unit of Shanghai Scicence Academy (Shanghai, China), Cells were grown in DMEM supplemented with 10% FBS and 1% NEAA in a humidified 5% CO2 atmosphere at…show more content…
10 μl FITC-conjugated Annexin V was added according to the manufacturer’s protocol. After incubation for fifteen minutes at room temperature in the dark, 10 μl of PI was added and following 200μl binding buffer was added and samples were immediately analyzed using FACSCalibur flow cytometer (BD Bioscience, USA). 1×104 cells were collected and analyzed with CellQuest software (BD Bioscience, USA). Apoptotic cells are expressed as a

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